Bacteriophage T4 Gene 44 DNA Polymerase Accessory Protein

3 downloads 95 Views 2MB Size Report
synthesis by T7 DNA polymerase (Bedinger and Alberts,. 1983; Nossal and Alberts, ... HCI, 0.2% phenol) on a Beckman 121 M amino acid analyzer. Several peptides from ..... R., George, D. G., Blomquist, M. C. and Johnson. G. C. (1983)Atlas ...
Vol. 259, No. 24, Issue of December 25,pp. 15425-15432,1984 Printed in U.S .A.

THEJOURNALOF BIOLOGICAL CHEMISTRY

Bacteriophage T4 Gene 44 DNA Polymerase Accessory Protein SEQUENCES OF GENE 44 AND ITSPROTEIN PRODUCT* (Received for publication, December 27, 1983)

Eleanor K. SpicerSS, Nancy G. NossalV, andKenneth R. Williams# 11 From the $Department of Molecular Biophysicsand Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06510 and the llhboratory of Biochemical Pharmacology,National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, Bethesda, Maryland 20205

Bacteriophage T4 gene 44 protein is a DNA polymerase accessory protein which is required for T4 DNA replication. We have isolated the gene for 44 protein from a previously constructed X-T4 hybrid phage (Wilson, G. G., Tanyashin, V. I., and Murray, N. E. (1977) Mol. Gen. Genet. 156, 203-214). We report here the nucleotide sequence of gene 4 4 and about 60 nucleotides 5’ upstream from its coding region,which is immediately adjacent to gene45. We have alsopurified 44 protein from T4-infected cells and submitted it to extensive protein chemistry characterization. Thus, considerable portions of the protein sequence predicted from the DNA sequence were confirmed by direct protein sequencingof peptides or by matching amino acid compositions of purified peptides. A total of 84%of the predicted amino acids was confirmed by the protein data. These studies indicate that gene 44 codes for a polypeptide containing 319 amino acids, with a calculated M. = 35,371. The coding region of gene 44 is preceded by a potential regulatory region containing sequences homologous to the Escherichia coli (-10) RNA polymerase binding region and to a conserved sequence at -25 to -30 found in other T4 middle genes. In addition, there are sequence similarities in the translation initiation regions of genes 44, 45, and rIIB, all of which are subject to regulation by regA protein.

The product of bacteriophage T4 gene 44 is a DNA polymerase accessory protein that plays an essential role in T4 DNA replication (Epstein et al., 1963). In vitro studies have demonstrated that efficient T4 DNA synthesis can be obtained by the coordinate action of seven T4 proteins, the products of gene 43 (DNA polymerase), gene 32 (ssDNA’ binding protein), genes 44,45, and 62 (polymerase accessory proteins), and genes 41 and 61 (RNA priming proteins) (Liu et al., 1979; Silver and Nossal, 1979; Nossal and Alberts, 1983). Addition *We are grateful to Dr. William Konigsberg (United States Public Health Service Grant GM112607) for his continued support of this work. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 5 Recipient of United States Public Health Service National Institutes of Health Grant GM3019101. I( Recipient of United States Public Health Service National Institutes of Health Grant GM31539 and National Science Foundation Grant PCM-8302908. The abbreviations used are: ssDNA, single-stranded DNA; dsDNA, double-stranded DNA kb, kilobase pair(s); bp, base pair(s); ATPyS, adenosine 5’-O-(thiotriphosphate);HPLC, high-performance liquid chromatography.

of the threeDNA polymerase accessory proteins to anin vitro synthesis reaction using primed ssDNA templates increases both the rate and theaverage length of the product made by T4 DNA polymerase (Alberts et al., 1977; Piperno and Alberts, 1978). The accessory proteins are also required for efficient strand displacement synthesis, using nicked dsDNA (Liu et al., 1979; Silver and Nossal, 1979; Nossal and Peterlin, 1979). In addition, the 44/62 and 45 proteins increase the rate of hydrolysis of dsDNA by the 3‘ to 5’ exonuclease activity of T4 DNA polymerase (Alberts et al., 1980; Venkatesan and Nossal, 1982; Bedinger and Alberts, 1983). Gene 44 protein and gene 62 protein form a multisubunit complex which exhibits an ATPase activity stimulated by ssDNA (Piperno and Alberts, 1978) or by duplex DNA with a protruding5’ single strand, analogous to theDNA structure at a primer-template junction(Munn andAlberts, 1983). This ATPase activity, which is enhanced by 45 protein, is required for the increased rate and extentof DNA synthesis observed upon the addition of accessory proteins to an i n vitro DNA synthesis reaction (Piperno and Alberts, 1978; Huang et al., 1981). It has been proposed that the accessory proteins hydrolyze ATP during the formation of a complex with T4 DNA polymerase (Alberts et al., 1980; Newport et al., 1980). This complex appears to stabilize the association of polymerase with DNA, acting as a “sliding clamp” that reduces the rate of dissociation of polymerase from the template (Alberts et al., 1980; Huang et al., 1981; Roth et al., 1982; Venkatesan and Nossal, 1982; Bedinger and Alberts, 1983). Maintenance of this complex requires periodic ATP hydrolysis as demonstrated by the effect of ATPyS on DNA synthesis in vitro (see Nossal and Alberts, 1983). All of these functions of the T4 accessory proteins apparentlyaredependent upon specific protein:protein interactions, since these proteins have no effect on the rate of DNA synthesis by T7 DNA polymerase (Bedinger and Alberts, 1983; Nossal and Alberts, 1983) or even on the exonuclease activity of an amber fragment of T4 DNA polymerase, amB22, which lacks the carboxyl-terminal 20% of the wild-type enzyme (Venkatesan and Nossal, 1982). As an important step in a continuing study of the structure-function relationships in this multiprotein complex, we have determined the nucleotide sequence of the regulatory and coding regions of gene 44 by DNA sequencing of the cloned gene and analysis of purified 44 protein. MATERIALS ANDMETHODS

Bacterial Strains and Bacteriophage-The Escherichia coliK12 derivative JM103 (A(&, pro), thi, strA, supE, endA,sbcB15, F’ traD36, proAB, lacIq ZAM15) described by Messing et al. (1981) was obtained from Bethesda Research Laboratories. Phage M13 derivatives mp7, mp8, and mp9 (Messing et al. 1981; Messing and Vieira,

15425

15426

T4 Gene 44 DNA Polymerase Accessory Protein

943 regA 962 rl45 1982) wereobtained from Bethesda Research Laboratories. .” -9 4 4 .” T4 DNA Phage X806-17 (Wilson et al., 1977) wasgenerously provided by G. ECDRI PI1 I “8”d m m dm restrlctlon sltes G. Wilson(New England Biolabs). X806-17was grown on E. coli 4 L 1 4 ED8689 (Wilson et al., 1977) in Nz Amine media (Kraft Co.) using a MAP UNITS I I 1 29 0 30 0 31 0 3:o (Kb) protocol for X propagation described by Williams and Blattner(1979). Enzymes and Biochemicals-Restriction endonucleases were puri chased from Bethesda Research Laboratories and New England BioX-806-17 ’ x (56KbT4 labs. T4 DNAligase and deoxy- and &deoxyribonucleotides were ~nsarll purchased from P-L Biochemicals. E. coli DNApolymerase I Klenow fragment was purchased from Boehringer Mannheim. [CY-~’P]~ATP - rnP8 m m 7 T 4 DNA 1 subclones was purchased from New England Nuclear and Amersham Corp. P11110, Cloning of Gene 44-T4 cytosine-containing DNA carrying gene 44 was obtained from the X-T4 recombinant phage X806-17 (Wilson et FtG. 1. DNA replication regionof the T4 genome. The map al. 1977), which contains all of gene 44. DNA restriction fragments units refer to the distance from the rIIA/rIIB junction (Kutter and were purified from X806-17 DNA and inserted into plasmid pBR322. Ruger, 1983). X806-17 (Wilson et al., 1977) contains a 5.6-kb EcoRI Plasmid DNA was prepared by the method of Birnboim and Doly fragment of T4 DNA from which DNA containing all of gene 44 was (1979). Restriction fragments carrying gene 44 were isolated from obtained. PstI andHindIII restriction fragments (indicated by dotted phage and plasmid DNA by polyacrylamide gel electrophoresis, fob lines) were cloned into pBR322 or M13 vectors, as shown. lowed by electroelution of gel slices into dialysis bags (McDonnell et al., 1977). Restriction fragments were subcloned into M13 mp7, mp8, or mp9 replicative form DNA, using standard cloning techniques product influences the level of expression of genes 44,45,62, (Maniatis et al., 1982). Recombinant phage were selected either on and others (see below for discussion). Genetic analysis has the basis of blue plaque to white plaque transition (Messing et al., revealed the orderof these 5 genes (for review see Woods and 1981) or by filter hybridization of phage plaques (Grunstein and Revel, 1976), andrestrictionanalysisand cloning studies Hogness, 1975). When blunt-ended fragments were cloned, a sufficient number of recombinant phage were isolated so that they con- (Wilson et al., 1977; Mattson et al., 1977; Niggemann et al., 1981) have further defined their physical location on the T4 tained fragments insertedin both orientations. DNA Sequencing-All sequencing experiments employed the genome. The clustering of the four replication protein genes method of Sanger et a!. (1977), utilizing recombinant M13 phage may facilitate a coordinate assembly of their gene products carrying inserts of T4 DNA. Growth of M13 on JM103, purification of ssDNA templates, and the dideoxy chain-terminating reaction into a functional replication complex, or it may be related to conditions were carried out as previously described (Spicer et al., the mechanismscontrolling their temporal expression. 1982). The 17’-mer, 5‘-GTAAAACGACGGCCAGT-3’ (Duckworth et al., 1981), which was used as a primer in the sequencing reactions, Subcloning and Sequence Analysis of Gene 44 was the generous gift ofM. Gait, M.R.C., Cambridge. Sequencing Cytosine-containing T 4 DNA carrying gene 44 was isolated reactions were analyzed on 8% (for nucleotides 1-200) and 5% (nucleotides 200-450) polyacrylamide, 7 M urea gels, which were dried from X806-17 andfrom a previously constructedhybrid ontoWhatman 3MM paper prior to autoradiography on Dupont plasma of pBR322 and T4 DNA, pAR101. As shown in Fig. Cronex 4 film. 1, pARlOl contains a 1.6-kb HindIII fragment of T 4 DNA Purification of 44 Protein”T4 gene 44 protein was purified as a complex with 62 protein by the procedure described previously (Nos- which carries all of gene 45 and approximately 40% of gene sal, 1979). Four mg of the complex were carboxamidomethylated and 44 (Spicer et al., 1982). The remaining 60% of gene 44 is then subjected to automated Edmandegradation on aBeckman 890C located within an 1100-bp HindIIJIPstI fragment of T4 conSequencer. Amino acid analyses of 44 protein and itstryptic peptides tained in X806-17 (Wilson et al., 1977). DNA containing g44 were determined (after hydrolyzing aliquots for 16 h at 115 “C in 6 M HCI, 0.2%phenol) on a Beckman 121 M amino acid analyzer. Several was isolated by Hind111 digestion of pARlOl or HindIII and peptides from 44 protein were sequenced by the manual procedure PstI digestion of X806-17 and subsequently inserted into M13 described by Tomita et al. (1978) (for peptides containing less than cloning vectors as shown schematically in Fig. 1. M13-T4 15 amino acids) or by solid-phase sequencing on a Sequemat mini-15 recombinant phage were identified by plaque filter hybridiSequencer equipped with a P-6 autoconverter (L‘Italien and Laursen, 1982; L’Italien and Strickler, 1982). In the latter case, the peptides zation to the appropriate32P-labeledrestriction fragments of were first immobilized to aminopolystyrene (Sequemat) using the pARlOl or X806-17 (Grunstein and Hogness, 1975). water-soluble carbodiimide procedure described previously (L’Italien Restriction enzyme analysis of the T4DNA spanning gene and Strickler, 1982). Phenylthiohydantoin derivatives were identified 44 indicated thepresence of 1AluI site to the right of gene 44 by amino acid analysis after hydrolysis with hydriodic acid for 18 h and 1 RsaI site, 1 Sau3A site, and 1 HindIII site within gene at 130 “C (Smithies et al., 1971) or by high-performance liquid chromatography on an Altex Ultrasphere RP-18 column (L’Italien and 44, as shown in Fig. 2. To facilitate nucleotidesequence Strickler, 1982). Additional details on the isolation and sequencing analysis of the 1.5-kb region spanning 844, restriction fragof gene 44 peptides are given in the Miniprint Supplement at theend ments of 300-500-bp lengths generated by these restriction of this paper. enzymes were cloned into M13 mp7, mp8, and mp9. These

-

~

recombinant phage ssDNAs served astemplates for DNA sequence analysis by the method of Sanger et al. (1977). Fig. The T4 genes coding for DNA polymerase (gene 43) and 2 summarizes the restriction fragments which were cloned the polymerase accessory proteins (genes 44,45, and 62) mapinto M13 and the direction and lengthof sequence determined within a 6.5-kb region of the T4 genome, as shown inFig. 1. from each clone. DNAsequences were determined for the Included inthiscluster of genes is the regA gene whose regionsoverlapping each of therestrictionsites used for subcloning, except for the HindIII site. In the case of the Portions of this paper (including part of “Results,” Tables 1-111, and Figs, 1-3) are presented in miniprint at the end of this paper. Hind111 site, the continuity of the DNA sequence (i.e. the Miniprint is easily read with the aid of a standard magnifying glass. absence of an undetected additional HindIII site) was con(described below). The Full size photocopies are available from the Journal of Biological firmed by proteinchemistrydata Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request DOC- results of the sequence analysis are given in Fig. 3, which ument No. 83M-3668, cite the authors, and include a check or money presents the complete nucleotide sequence of the structural order for $2.80 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from gene for 44 protein and the derived amino acid sequence of 44 protein. Waverly Press. RESULTS AND DISCUSSION’

T4 Gene 44 DNA Polymerase Accessory Protein

15427

plement) indicated a 3.6(& 0.6):l molar ratio of 44 protein to 62 protein and identified the first 30 amino acids in 44 protein, indicated by solid underliningin Fig. 3. In addition, sequence analysis enabled the unambiguous identification of the translational start and the correct reading frame of gene 44. In order to obtain additional protein chemistry data on peptides derived from44 protein, 44 protein was purified by subjecting the carboxamidomethylated 44 protein-62 protein complex to Sephacryl S-300 gel filtration (Fig. 4).The purified 44 protein was then digested with trypsin and theresulting peptides were separated by HPLC (Fig. 1 in the Miniprint Section). Additional peptides were isolated by digesting some incompletely I I I 1 30.0 30.5 31.0 31.5 resolved tryptic peptides with chymotrypsin and then subMAP UNITS (Kb) jecting the resulting peptides to HPLC (Figs. 2 and 3 in the FIG. 2. Restriction enzyme map of the T4 region spanning Miniprint Supplement). The amino acid compositions of all gene 44. DNA fragments purified from pARlOl or T4M13 hybrid these peptides are summarized in Tables I1 and I11 in the phage were cleaved byRsaI, Sau3A, or Ah1 and cloned into M13 mp8 or mp8.The wauy lines indicate the length and direction of nucleotide Miniprint Supplement. In addition, several of these peptides sequence determined from the M13-T4 recombinants. *indicatesthe were partially sequencedto further confirm the DNA sequence sole AluI site used inthe cloning procedures; there are three additionalof gene 44. The results of these studies are summarized in AluI sites that lie between 30.0 and 31.0-kb map units, which are not Fig. 3, where the solid underlining indicates portions of the shown. 44 protein sequence confirmed by direct amino acid sequencing, and thedotted lines indicate regions that were confirmed by matching amino acid compositions of purified peptides with those predicted from the DNA. In all, a total of 84% of the amino acids predicted from the DNAsequencewere confirmed by the protein data, and no inconsistencies were observed. The results derived from the sequence data demonstrate that 44 protein is composed of 319 amino acids and has a molecular weight of 35,731. Previous estimates of the size of 44 protein, determined by polyacrylamide gel electrophoresis, were 34,000 (Barry and Alberts, 1972) and 32,000 (Nossal, 1979). The predicted and experimentally determined amino acid compositions of 44 protein are given in Table I. There is, in general, excellent agreement between the experimentally determined values and thesum of the predicted residues, with the exception of the number of isoleucine residues, where 8 more residues are predicted from the DNA sequence than the amino acid analysis shows. This can be rationalized by the fact that there are16 X-isoleucine-Xlinkages (whereX stands for isoleucine, leucine, or valine), which are cleaved with an efficiency of only about 50%, using the acid hydrolysis conI

9 44

I

I

U P

I

66K

43K

1

2

3

-

31K-

-44P

-62P

18K14K-

Protein Chemistry Studies of 44 Protein Gene 44 protein and 62 protein co-purify from T4-infected cells as a tightly associated multisubunit complex. The molar ratio of 44 protein to 62 protein in this complex appears to be variable; Barry and Alberts (1972) purified a complex with a 44 protein:62 protein molar ratio of 42, while Nossal (1979) observed a ratio of 5.51. In order to determine the stoichiometry of the complex moreaccurately and to determine the NH1-terminal sequence of44 protein, carboxamidomethylated complex was subjected to automatic (liquid phase) sequencing. This analysis (see Table I in the Miniprint Sup-

IOKFRACTION NUMBER

FIG. 4. Purification of T4 protein 44 (44P). Approximately 6 mg ofgene 44-62 proteincomplexwerecarboxamidomethylated, dialyzed uersus 1 m M NaHCOs. and then lyophilized priorto dissolvingin 1 ml of 10 mM Tris, pH 8.0, 6 M guanidinehydrochloride (buffer A). The sample was then appliedto a column (1.5 X 195 cm) of Sephacryl S-300 equilibrated with buffer A and run at 15 ml h". Fractions (2 ml) were collected, their absorbance at 280 nm read, and pooled as indicated on the left above. The right side of the above figure shows a 17.5% polyacrylamide sodium dodecyl sulfate gel of the purified 44 (lane 2) and 62 (lane 3) proteins.

T4 Gene 44 DNA Polymerase Accessory Protein

15428

ditions given under “Materials and Methods.” As shown in Table I, 44 protein contains 7 cysteines, 10 methionines, and is rich in the hydrophobic residues isoleucine, leucine, and valine (23%).

p-structure and 17% a-helix while the COOH-terminal onethird contains 12%&structure and56% a-helix. The 39 basic and 41 acidic residues are evenly distributed throughout the polypeptide chain, with the exception of a small cluster of acidic residues at positions 162-167. Secondaty Structureof 44 Protein Comparisons of DNA binding proteins have indicated similarities in secondary structure which are postulated to be The secondary structure of 44 protein predicted by the empirical method of Chou and Fasman (1978) wasdetermined related to nucleotide and DNA binding. The supersecondary by computer program analysis (Cohen, 1979) and is shown in structure P-a-p-a-p has been correlated with a nucleotide fold Fig. 5. Based on this analysis, 44 protein is predicted to have in anumber of proteins (Raoand Rossmann, 1973; Rossmann 32% a-helix, 23% p-structure, and 45% random coil. Although et al., 1974). The predicted secondary structure of 44 protein the distribution of ordered regions (a-helix and P-structure) contains a sequence of p-a-@-astructures (residues 81-128) in 44 protein i s relatively uniform (Fig. 5), the distribution of and, in addition, contains two antiparallel sequential p-p a-helical and P-sheet regions is asymmetric. The NHz-termi- dimer structures (at residues 150-177 and 272-287). The ,O-P nal one-third of 44 protein is predicted to contain about 42% dimers have been proposed to form a “polypeptide double helix” which may play a role in nucleic acid binding (Carter TABLE I and Kraut, 1974; Chou and Fasman, 1978). Amino acid composition of 44 protein Residue

Ala Arg Asn

No. medicted

No. determined

26

27.0 16.9

14 13 21

ASP

7

CYS Gln Glu GIY His Ile Leu

11 20 17

8 32 24 25 10

LYs Met Phe Pro Ser Thr Trp

13

TY~ Val

ND, not determined.

11 22 16 2 9 18

36.7

5.8 34.3 18.6 8.8 24.0 22.3

26.9 9.7 12.8 13.0

23.9 16.3

ND” 9.3 16.4

Homologies Between 44 Protein and ATPases As noted earlier, the 44 protein-62 proteincomplex exhibits a ssDNA-dependent ATPaseactivity. To date, it hasnot been possible to renature the individual subunits and todetermine directly which of the two proteins exhibits the ATPase activity. Recently Walker et al. (1982) reported amino acid homologies in six ATP-binding enzymes which they suggest may be related to the presence of a common adenine nucleotide binding fold. Comparison of those conserved sequences with the sequence of44 protein reveals one region of similarity which is illustrated in Fig. 6. The sequence Gly-Thr-Gly-LysThr (residues 53-57 in 44 protein) is found in E. coli ATP synthase a-subunit, andhomologous or similar residues (e.g. serine and threonine) are found in bovine and E. coli ATP synthase&subunits, myosin, adenylate kinase, and RecA protein. To evaluate the significance of this sequence homology, the sequence of26 residues spanning 39 to 64of44 protein was compared to the Protein Sequence Database of Dayhoff et al. (1983). In comparing 2,377 sequences (contain-

-

FIG. 5. Predicted secondary structure of 44 protein. %,M, and denote a-helical, 8-structure,and random coil residues, respectively. @-Structureturns are denoted by chain reversals. The + and signs indicate charged residues, and thenumbers refer to residues a t conformational boundaries.

-

L z

15429

T4 Gene 44 D N A Polymerase Accessory Protein PROTEIN

8

T4 44 P r o t e i n E. c o l i

ATPase-

"

Pro

Set

Pro Gly Thr Gly Lys Thr Thr

Gly

Asp

Arg

Cly Gly Pro Gly

AdenylateKinase, porcine

Gly

Thr

Ser

Gly Lys

Thr

Ala

Val

Ala

Lye

Leu Gln

Ala

I l eCys

Gly

Lys

#178 -

Arg

8312

E. 1c6o7l i RecA p r o t e i n

"

m y o s i n ,r a b b i t

A m

T7 gene 4 p r o t e i n

Phe Val

Thr

- 60 $169 Glu - 180 115 - 26 150

-

1 2,3

78

4

190

5

323

6

FIG. 6. Amino acid sequences in 4 4 protein homologous to sequences in five ATP-binding proteins Walker et d.,1 9 8 2 ) . Proteins are aligned to indicate maximum homology. Boxed residues are homologous

(see

or very similar (eg. Ser = Thr), andcircled residues indicate similar amino acids in three or four of the five proteins. Sequences are from: I , Gay and Walker (1981); 2, Pai et al. (1977); 3, Heil et al. (1974); 4, Sancar et al. (1980); 5, Walker et al. (1980); 6,Dunn and Studier (1981).

ing 371,232 segments of 26 amino acids),4 additional proteins3 were found to containsequences similar to the Gly-Thr-GlyLys-Thr sequence. Only 1of the 4 proteins, T 7 gene 4 protein (shown in Fig. 6) is known to have a nucleotide triphosphate binding a~tivity.~ In terms of the overall homology of the 26 residues of44 protein, seven proteins exhibited sequences with higher matrix (homology) scores than adenylate kinase, and ATP synthase and recA exhibited relatively low homology scores. Thus, if there is any relatednessof 44 protein to ATP binding proteins it is limited to these few amino acids shown in Fig. 6.

T A T A A T

I T A A T G C T T C

5

6

Expression of Gene 44 Transcription-Gene 44 belongs to the pre-replicative middle class of T 4 genes which are expressed soon after phage infection (see Rabussay, 1982). Early studies suggested that genes 45, 44, and 62 (see Fig. 1) were co-transcribed from a single promoter lying upstream of gene 45 (Stahl etal., 1970; HerculesandSauerbier, 1973). b w e v e r , morerecently Bowles and Karam (1979)isolateda mutation mapping in front of44 protein which resulted in overproduction of44 protein and 62 protein but did not affect 45 protein production, suggesting there is a regulatory site in frontof gene 44. These studies further indicate that the mutation led to increasedtranscriptionratherthantranslation of gene 44 mRNA. They, therefore, proposed that a promoter lies upstream of gene 44 from which polycistronic gene44-62 mRNA synthesis is initiated. Transcription of T 4 early and middle promoters is carried out by an altered formof the host E.coli RNA polymerase, whose recognition and binding signals are not, as yet, well characterized. Examination of the few presumed T4 early and middle promoters which have been sequenced indicates they have strong homologies to the (-10) RNA polymerase binding region of E. coli promoters butonly weak homologies to the E. coli (-35) regions (Brody et al., 1983). In addition, the T 4 promoters have a conserved sequence a t -25 to -30 of unknown function, which may play a role in RNA polymerase recognition or may function in n o t regulation (see Brody et al., 1983). As shown in Fig. 7, the potential regulatory region of gene 44 contains sequences The four proteins are genome polyprotein of poliovirus, T7 gene 4 protein, E. coli transposase repressor, and histidine permease inner

membrane receptor protein P of Salmonella typhimuriurn. T7 gene 4 protein is both a primase and a helicase, and itexhibits a nucleoside triphosphatase activity (Matson, S. W., and Richardson, C. C. (1983) J. Biol. Chern. 258,11165-11173; Matson, S. W., Tabor, S., and Richardson, C. C . (1983) J. Biol. Chern. 258, 14017-14024).

5'